Bone remodeling is a continuous process that maintains skeletal integrity through the coordinated activities of two cell populations: osteoclasts, which are responsible for bone resorption (osteolysis), and osteoblasts, which are responsible for bone formation. In order to maintain bone homeostasis, the activity of osteoclasts and osteoblasts must be tightly regulated. Dysregulation of osteoclast activity is associated with several forms of human disease, including osteolytic bone loss due to increased osteoclast activity and osteopetrosis (excessive bone accumulation) due to decreased osteoclast activity. Osteolytic bone loss may also be caused by cancer metastasis to bone. Breast, lung, and prostate cancer are the most common malignancies in adults, and they are also the most common tumor types to metastasize to bone. Kidney, thyroid, and melanoma are other common forms of cancer that metastasize to bone. Tumor cells often secrete osteoclast-activating factors, including the colony stimulating factor 1 ligand (referred to herein as CSF1; also referred to in the art as MCSF and MGC31930) and the receptor activator of NF-kB ligand (RANKL), that stimulate osteoclast activity and induce osteolytic bone loss. As a result, bone metastasis is associated with fractures, pain, spinal cord compression, and hypercalcemia. Therefore, therapeutic agents that treat osteolytic bone loss, inhibit metastasis, and/or prevent metastasis-induced osteoclast activation would be expected to offer significant clinical benefits to patients.
The colony stimulating factor 1 receptor (referred to herein as CSF1R; also referred to in the art as FMS, FIM2, C-FMS, and CD115) is a single-pass transmembrane receptor with an N-terminal extracellular domain (ECD) and a C-terminal intracellular domain with tyrosine kinase activity. Ligand binding of CSF1 or the interleukin 34 ligand (referred to herein as IL34; also referred to in the art as C16orf77 and MGC34647) to CSF1R leads to receptor dimerization, upregulation of CSF1R protein tyrosine kinase activity, phosphorylation of CSF1R tyrosine residues, and downstream signaling events. Both CSF1 and IL34 stimulate monocyte survival, proliferation, and differentiation into macrophages. However, IL34 was discovered recently, and its overall functions have not been fully established.
Dysregulation of CSF1R activity may result in an imbalance in the levels and/or activities of macrophage and osteoclast cell populations, which may lead to disease-associated pathology. Based on their known and suspected contributions to human disease, both CSF1R and CSF1 have been identified as potential therapeutic targets for osteolytic bone loss and cancer. Indeed, CSF1R and CSF1 antagonists such as antibodies directed against CSF1R or CSF1 (see e.g., WO 2007/081879), antisense- and siRNA-mediated silencing of CSF1R or CSF1 expression (see e.g., WO 2007/081879), soluble forms of the CSF1R (see e.g., WO 2007/081879), and small molecule inhibitors of CSF1R tyrosine kinase activity (see e.g., Ohno et al., Molecular Cancer Therapy 5(11): 2634-2643 (2006); Murray et al., Clinical & Experimental Metastasis 20:757-766 (2003); and Conway et al., PNAS 102(44):16078-16083 (2005)), have been proposed for targeting osteolytic bone loss. Despite the proposed utility of such CSF1R and CSF1 antagonists, there remains a need in the art for the identification of additional agents with a demonstrated ability to inhibit disease pathology in vivo, such as osteolytic bone loss, cancer metastasis, cancer metastasis-induced osteolytic bone loss, and tumor growth.
To date, we are not aware of any demonstration of a CSF1R ECD fusion molecule shown to be effective at treating osteolytic bone loss, cancer metastasis, cancer metastasis-induced osteolytic bone loss, or tumor growth in vivo. As described herein, the inventors have found that CSF1R ECD fusion molecules of the invention inhibit osteolytic bone loss in vivo models, including osteolytic bone loss resulting from cancer metastasis to bone (See Examples 8 and 10). Furthermore, the inventors have also shown that CSF1R ECD fusion molecules inhibit cancer metastasis in vivo (See Example 9). In certain cancers, the inventors have found that CSF1R ECD fusion molecules directly inhibit primary tumor growth in vivo (See Example 9).
In addition, the inventors have found that certain of the CSF1R ECD fusion molecules of the invention exhibit improved properties, including improvements to therapeutically relevant properties. For example, the inventors have found that expression of CSF1R ECD fusion molecules in CHO cells results in more highly sialylated CSF1R ECD fusion molecules, which are more stable than such fusion molecules produced in 293-6E cells. Also, the inventors have found that a CSF1R ECD fusion molecule wherein the CSF1R ECD has the amino acid sequence of SEQ ID NO.:2 (amino acids 20-506 of the human CSF1R protein) binds the CSF1R ligands CSF1 and IL34 more tightly and more effectively inhibits monocyte growth in an in vitro assay than a full-length CSF1R ECD fusion molecule wherein the CSF1R ECD has the amino acid sequence of SEQ ID NO.:1 (amino acids 20-512 of the human CSF1R protein). Thus, this CSF1R ECD fusion molecule provides a particularly attractive therapeutic molecule.
Accordingly, the present invention provides methods and compositions for treating osteolytic disorders, including but not limited to, osteolytic bone loss. Osteolytic bone loss may include cancer metastasis-induced osteolytic bone loss. The present invention also provides methods and compositions for inhibiting cancer metastasis and inhibiting CSF1R-dependent tumor growth.
The invention includes a method of treating osteolytic bone loss in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners. The invention further includes a method of treating osteolytic bone loss in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the amino acid sequence of the CSF1R ECD consists of SEQ ID NO.:2, SEQ ID NO.:26, or SEQ ID NO.:1. In certain embodiments, the one or more fusion partners are selected from an Fc, albumin, and polyethylene glycol. In certain other embodiments, the one or more fusion partners of the are selected from an Fc alone, polyethylene glycol alone, and an Fc and polyethylene glycol. The invention further includes the method of treating osteolytic bone loss in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6 or SEQ ID NO.:5. The invention also includes the method of treating osteolytic bone loss in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6 or SEQ ID NO.:5, and wherein the CSF1R ECD fusion molecule is glycosylated or sialylated. The invention further includes the method of treating osteolytic bone loss in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the CSF1R ECD fusion molecule is produced from a CHO cell. The invention further includes the method of treating osteolytic bone loss in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the osteolytic bone loss is cancer metastasis-induced.
The invention includes a method of inhibiting cancer metastasis in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners. The invention further includes a method of inhibiting cancer metastasis in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the amino acid sequence of the CSF1R ECD consists of SEQ ID NO.:2, SEQ ID NO.:26, or SEQ ID NO.:1. In certain embodiments, the one or more fusion partners are selected from an Fc, albumin, and polyethylene glycol. In certain other embodiments, the one or more fusion partners of the are selected from an Fc alone, polyethylene glycol alone, and an Fc and polyethylene glycol. The invention further includes the method of inhibiting cancer metastasis in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6 or SEQ ID NO.:5. The invention also includes the method of inhibiting cancer metastasis in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6 or SEQ ID NO.:5, and wherein the CSF1R ECD fusion molecule is glycosylated or sialylated. The invention further includes the method of inhibiting cancer metastasis in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the CSF1R ECD fusion molecule is produced from a CHO cell. The invention further includes the method of inhibiting cancer metastasis in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, kidney cancer, thyroid cancer, melanoma, follicular lymphoma, carcinoma with p53 mutations, colon cancer, cardiac tumor, pancreatic cancer, retinoblastoma, glioblastoma, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, Kaposi's sarcoma, ovarian cancer, leukemia, acute leukemia, chronic leukemia, myelodysplastic syndrome polycythemia vera, multiple myeloma, Waldenström's macroglobulinemia, and heavy chain disease. The invention further includes the method of inhibiting cancer metastasis in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the cancer is breast cancer.
The invention includes a method of treating CSF1- and/or IL34-dependent tumor growth in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners. The invention further includes a method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the amino acid sequence of the CSF1R ECD consists of SEQ ID NO.:2, SEQ ID NO.:26, or SEQ ID NO.:1. In certain embodiments, the one or more fusion partners are selected from an Fc, albumin, and polyethylene glycol. In certain other embodiments, the one or more fusion partners of the are selected from an Fc alone, polyethylene glycol alone, and an Fc and polyethylene glycol. The invention further includes the method of treating CSF1- and/or IL34-dependent tumor growth in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6 or SEQ ID NO.:5. The invention also includes the method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6 or SEQ ID NO.:5, and wherein the CSF1R ECD fusion molecule is glycosylated or sialylated. The invention further includes the method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the CSF1R ECD fusion molecule is produced from a CHO cell. The invention further includes a method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the tumor is a solid tumor or a leukemic tumor. The invention further includes a method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the tumor is a solid tumor or a leukemic tumor, and wherein the solid tumor is selected from sarcoma and carcinoma.
The invention includes a method of treating osteolytic bone loss in a patient who has and/or has been diagnosed with osteolytic bone loss comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners. The invention further includes a method of treating osteolytic bone loss in a patient who has and/or has been diagnosed with osteolytic bone loss comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the amino acid sequence of the CSF1R ECD consists of SEQ ID NO.:2, SEQ ID NO.:26, or SEQ ID NO.:1. In certain embodiments, the one or more fusion partners are selected from an Fc, albumin, and polyethylene glycol. In certain other embodiments, the one or more fusion partners of the are selected from an Fc alone, polyethylene glycol alone, and an Fc and polyethylene glycol. The invention further includes the method of treating osteolytic bone loss in a patient who has and/or has been diagnosed with osteolytic bone loss comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6 or SEQ ID NO.:5. The invention also includes the method of treating osteolytic bone loss in a patient who has and/or has been diagnosed with osteolytic bone loss comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6 or SEQ ID NO.:5, and wherein the CSF1R ECD fusion molecule is glycosylated or sialylated. The invention further includes the method of treating osteolytic bone loss in a patient who has and/or has been diagnosed with osteolytic bone loss comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the CSF1R ECD fusion molecule is produced from a CHO cell. The invention further includes the method of treating osteolytic bone loss in a patient who has and/or has been diagnosed with osteolytic bone loss comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the osteolytic bone loss is cancer metastasis-induced.
The invention includes a method of inhibiting cancer metastasis in a patient who has and/or has been diagnosed with cancer metastasis comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners. The invention further includes a method of inhibiting cancer metastasis in a patient who has and/or has been diagnosed with cancer metastasis comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the amino acid sequence of the CSF1R ECD consists of SEQ ID NO.:2, SEQ ID NO.:26, or SEQ ID NO.:1. In some embodiments, the CSF1R ECD fusion molecule comprises SEQ ID NO.:2 (terminating at residue 506 of the human CSF1R ECD amino acid sequence) and excludes the last six C-terminal amino acid residues of SEQ ID NO:1 (corresponding to residues 507-512 of the human CSF1R ECD sequence). In such fusion molecules, any amino acid residues that follow the C-terminal residue of SEQ ID NO:2 do not begin with the amino acid sequence of residues 507-512 of SEQ ID NO:1 (THPPDE). Such fusion molecules may of course include the amino acid sequence THPPDE anywhere else in the amino acid sequence. In certain embodiments, the one or more fusion partners are selected from an Fc, albumin, and polyethylene glycol. In certain other embodiments, the one or more fusion partners of the are selected from an Fc alone, polyethylene glycol alone, and an Fc and polyethylene glycol. In some embodiments, one or more fusion partners is joined to the CSF1R ECD by a linker. In some such embodiments, the linker is a peptide consisting of the amino acid sequence glycine-serine. In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the fusion molecule is glycosylated and/or sialylated. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells. The invention further includes the method of inhibiting cancer metastasis in a patient who has and/or has been diagnosed with cancer metastasis comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises or consists of SEQ ID NO.:6 or SEQ ID NO.:5. In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the fusion molecule is glycosylated and/or sialylated. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells. The invention also includes the method of inhibiting cancer metastasis in a patient who has and/or has been diagnosed with cancer metastasis comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises or consists of SEQ ID NO.:6 or SEQ ID NO.:5, and wherein the CSF1R ECD fusion molecule is glycosylated or sialylated. In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells. The invention further includes the method of inhibiting cancer metastasis in a patient who has and/or has been diagnosed with cancer metastasis comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the CSF1R ECD fusion molecule is produced from a CHO cell. The invention further includes the method of inhibiting cancer metastasis in a patient who has and/or has been diagnosed with cancer metastasis comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule as described previously, and wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, kidney cancer, thyroid cancer, melanoma, follicular lymphoma, carcinoma with p53 mutations, colon cancer, cardiac tumor, pancreatic cancer, retinoblastoma, glioblastoma, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, Kaposi's sarcoma, ovarian cancer, leukemia, acute leukemia, chronic leukemia, myelodysplastic syndrome polycythemia vera, multiple myeloma, Waldenström's macroglobulinemia, and heavy chain disease. The invention further includes the method of inhibiting cancer metastasis in a patient who has and/or has been diagnosed with cancer metastasis comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule as described previously, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the cancer is breast cancer.
The invention includes a method of treating CSF1- and/or IL34-dependent tumor growth in a patient who has and/or has been diagnosed with CSF1- and/or IL34-dependent tumor growth comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners. The invention further includes a method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient who has and/or has been diagnosed with CSF1- and/or IL34-dependent tumor growth comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the amino acid sequence of the CSF1R ECD consists of SEQ ID NO.:2, SEQ ID NO.:26, or SEQ ID NO.:1. In some embodiments, the CSF1R ECD fusion molecule comprises SEQ ID NO.:2 (terminating at residue 506 of the human CSF1R ECD amino acid sequence) and excludes the last six C-terminal amino acid residues of SEQ ID NO:1 (corresponding to residues 507-512 of the sequence). In such fusion molecules, any amino acid residues that follow the C-terminal residue of SEQ ID NO:2 do not begin with the amino acid sequence of residues 507-512 of SEQ ID NO:1 (THPPDE). Such fusion molecules may of course include the amino acid sequence THPPDE anywhere else in the amino acid sequence. In certain embodiments, the one or more fusion partners are selected from an Fc, albumin, and polyethylene glycol. In certain other embodiments, the one or more fusion partners of the are selected from an Fc alone, polyethylene glycol alone, and an Fc and polyethylene glycol. In some embodiments, one or more fusion partners is joined to the CSF1R ECD by a linker. In some such embodiments, the linker is a peptide consisting of the amino acid sequence glycine-serine. In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the fusion molecule is glycosylated and/or sialylated. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells. The invention further includes the method of treating CSF1- and/or IL34-dependent tumor growth in a patient who has and/or has been diagnosed with CSF1- and/or IL34-dependent tumor growth comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises or consists of SEQ ID NO.:6 or SEQ ID NO.:5. In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the fusion molecule is glycosylated and/or sialylated. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells. The invention also includes the method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient who has and/or has been diagnosed with CSF1- and/or IL34-dependent tumor growth comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the one or more fusion partners is an Fc, and wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises or consists of SEQ ID NO.:6 or SEQ ID NO.:5, and wherein the CSF1R ECD fusion molecule is glycosylated or sialylated. In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells. The invention further includes the method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient who has and/or has been diagnosed with CSF1- and/or IL34-dependent tumor growth comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the CSF1R ECD fusion molecule comprises a CSF1R ECD and one or more fusion partners, and wherein the CSF1R ECD fusion molecule is produced from a CHO cell. The invention further includes a method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient who has and/or has been diagnosed with CSF1- and/or IL34-dependent tumor growth comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule as described previously, wherein the tumor is a solid tumor or a leukemic tumor. The invention further includes a method of method of treating CSF1- and/or IL34-dependent tumor growth in a patient who has and/or has been diagnosed with CSF1- and/or IL34-dependent tumor growth comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule as described previously, wherein the tumor is a solid tumor or a leukemic tumor, and wherein the solid tumor is selected from sarcoma and carcinoma.
The invention includes a CSF1R ECD fusion molecule comprising a CSF1R ECD and one or more fusion partners, wherein the amino acid sequence of the CSF1R ECD comprises SEQ ID NO.:2 and excludes the last six C-terminal amino acid residues of SEQ ID NO.:1. In such fusion molecules, any amino acid residues that follow the C-terminal residue of SEQ ID NO:2 do not begin with the amino acid sequence of residues 507-512 of SEQ ID NO:1 (THPPDE). Such fusion molecules may of course include the amino acid sequence THPPDE anywhere else in the amino acid sequence. In some such embodiments, the CSF1R ECD consists of SEQ ID NO:2.
In certain embodiments, the one or more fusion partners are selected from an Fc, albumin, and polyethylene glycol. In certain other embodiments, the one or more fusion partners of the are selected from an Fc alone, polyethylene glycol alone, and an Fc and polyethylene glycol. In some embodiments, the fusion molecule comprises a linker between the CSF1R ECD and one or more fusion partners. In some such embodiments, the linker is a peptide consisting of the amino acid sequence glycine-serine. In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the fusion molecule is glycosylated and/or sialylated. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells.
The invention also includes a CSF1R ECD fusion molecule comprising a CSF1R ECD and an Fc, wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises SEQ ID NO.:6. The invention further includes the CSF1R ECD fusion comprising a CSF1R ECD and an Fc, wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6. In some such embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the fusion molecule is glycosylated and/or sialylated. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells.
The invention further includes a CSF1R ECD fusion molecule comprising a CSF1R ECD, an Fc, and polyethylene glycol, wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises SEQ ID NO.:6. The invention further includes a CSF1R ECD fusion molecule comprising a CSF1R ECD, an Fc, and polyethylene glycol, wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises SEQ ID NO.:6, and wherein the fusion molecule is glycosylated or sialylated. In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells.
The invention also includes a CSF1R ECD fusion molecule comprising a CSF1R ECD, an Fc, and polyethylene glycol, wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6. The invention also includes a CSF1R ECD fusion molecule comprising a CSF1R ECD, an Fc, and polyethylene glycol, wherein the amino acid sequence of the CSF1R ECD fusion molecule consists of SEQ ID NO.:6, and wherein the fusion molecule is glycosylated or sialylated. In some such embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells.
The invention further includes a pharmaceutical composition comprising any one of the above described CSF1R ECD fusion molecules and a pharmaceutically acceptable carrier.
The invention also includes a polynucleotide comprising a nucleic acid sequence that encodes any one of the above described CSF1R ECD fusion molecules. In some embodiments, the polynucleotide encodes a CSF1R ECD fusion molecule comprising a CSF1R ECD and one or more fusion partners, wherein the amino acid sequence of the CSF1R ECD comprises SEQ ID NO.:2 and excludes the last six C-terminal amino acid residues of SEQ ID NO.:1. The one or more fusion partners may include an Fc or albumin. The invention further includes a polynucleotide comprising a nucleic acid sequence that encodes a CSF1R ECD fusion molecule comprising a CSF1R ECD and one or more fusion partners, wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises SEQ ID NO.:6. In some embodiments, the amino acid sequence encoded by the polynucleotide of the invention comprises a signal peptide amino acid sequence. In some embodiments, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO:6 plus a signal peptide sequence, such as, for example, SEQ ID NO:16.
The invention further includes an expression vector comprising a polynucleotide as described above, encoding any one of the above described CSF1R ECD fusion molecules. The invention also includes a CHO cell comprising the expression vector comprising the polynucleotide encoding any one of the above described CSF1R ECD fusion molecules. For example, in some embodiments, the CHO cell comprises a vector comprising a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:6 plus a signal peptide amino acid sequence, such as, for example, SEQ ID NO:16.
The invention also includes a method of producing a CSF1R ECD fusion molecule. For example, in some embodiments, the method comprises: (a) culturing a CHO cell comprising the polynucleotide of any one of the above described CSF1R ECD fusion molecules in conditions such that the CSF1R ECD fusion molecule is expressed; and (b) recovering the CSF1R ECD fusion molecule. The invention further includes this method with the step of fusing polyethylene glycol to the CSF1R ECD fusion molecule.